The present invention is in the general field of the biosynthesis of biotin vitamers.
Biotin biosynthesis in Escherchia coli and Bacillus sphaericus has been studied at both the biochemical and molecular biological levels (DeMoll, 1996. In F. C. Neidhardt et al., (eds.) E. coli and Salmonella typhimurium: Cellular and Molecular Biology, Second edition ed., vol 1., pp. 704-709, ASM Press, Washington, D.C.; Perkins et al., In A. L. Sonenshein et al. (eds.), In Bacillus subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, pp. 319-334, American Society for Microbiology, Washington, D.C.; Eisenberg, 1987. In F. Neidhardt et al. (eds.), E. coli and Salmonella typhimurium, pp. 544-550. American Society for Microbiology, Washington, D.C.; Cronan, Cell 58:427-429, 1989, Izumi et al., Agric. Biol. Chem. 45:1983-1989, 1981; Gloeckler et al., Gene 87:63-70, 1990), although some steps and components in biotin synthesis remain to be elucidated (Ohshiro et al., Biosci. Biotech. Biochem. 58:1738-1741, 1994; Ifuku et al., Eur. J. Biochem. 224:173-178, 1994; Florentin et al., C. R. Acad. Sci. Paris 317:485-488, 1994; Birch et al., J. Biol. Chem. 270:19158-19165, 1995; Sanyal et al., Biochemistry 33:3625-3631, 1995). Several enzymes involved in the conversion of pimeloyl-CoA to biotin have been isolated and characterized from both of these bacterial species (Ploux et al., Biochem. J. 283:327-321, 1992; Izumi et al., Agric. Biol. Chem. 45:1983-1989, 1981; Eisenberg, supra, Huang et al., Biochemistry 34:10985-10995, 1995). KAPA synthase, the product of bioF, catalyzes the conversion of pimeloyl-CoA and alanine to 8-amino-7-ketopelargonic acid (KAPA). DAPA aminotransferase, the product of bioA, then transfers an amino group from a donor to KAPA yielding 7,8-diaminopelargonic acid (DAPA). Dethiobiotin synthetase (bioD) catalyzes the closure of the ureido-ring to produce dethiobiotin (DTB), and finally the product of bioB, biotin synthase, functions together with a number of other components including flavodoxin (Birch et al., supra; Ifuku et al., supra) S-adenosylmethionine (SAM) (Florentin, C. R. Acad. Sci. Paris 317:485-488, 1994; Ohshiro et al., supra; Sanyal et al., supra; Birch et al., supra) ferrodoxin NADP+ reductase (Birch et al., supra; Sanyal et al., Arch. Biochem. Biophys. 326:48-56, 1996) and possibly cysteine (Florentin, C. R. Acad. Sci. Paris 317:485-488, 1994; Birch et al., supra; Sanyal et al., supra) to convert dethiobiotin to biotin. The compounds KAPA, DAPA, DTB, and biotin are collectively or singly referred to as vitamers or biotin vitamers.
In E. coli the genes that encode these enzymes are located in two divergently transcribed operons, controlled by a single operator that interacts with the BirA repressor (Cronan, Cell 58:427-429, 1989). In B. sphaericus, the genes are located in two separate operons (Gloeckler et al., supra. The early steps of the pathway, those involved in the synthesis of pimeloyl-CoA, are less well understood (Ifuku et al., Eur. J. Biochem. 224:173-178, 1994; Sanyal et al., J. Am. Chem. Soc. 116:2637-2638, 1994). B. sphaericus contains an enzyme, pimeloyl-CoA synthetase (bioW) that converts pimelic acid to pimeloyl CoA (Gloeckler et al., Gene 87:63-70, 1990), (Ploux et al., Biochem. J. 287:685-690, 1992). E. coli lacks this enzyme and cannot use pimelic acid as an intermediate in biotin synthesis (Gloeckler et al., supra; Ifuku et al., Eur. J. Biochem. 224:173-178, 1994; Sanyal et al., J. Am. Chem. Soc. 116:2637-2638, 1994). E. coli contains two genes, bioC which is located in the bio operon and bioH which is unlinked to the other bio genes, that both appear to be involved in the early steps of biotin biosynthesis leading up to pimeloyl-CoA, but their exact roles are unknown (Eisenberg, supra; Lemoine et al., Mol. Micro. 19:645-647, 1996).
B. subtilis contains homologs of the E. coli and B. sphaericus bioA, bioB, bioD, and bioF genes. These four genes along with a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDB, and are followed by two additional genes, bioI and orf2 (Bower et al., J. Bacteriol. 178:4122-4130, 1996). bioI and orf2 are generally dissimilar to other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P450s and is able to complement mutations in either E. coli bioC or bioH (Bower et al., supra. Mutations in bioI cause B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants can be overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis (Bower et al., supra.
The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains, just downstream, a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus (Bower et al., supra. Analysis of a bioW-lacZ translational fusion indicates that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene. Strains deregulated for biotin synthesis can be engineered by replacing the promoter and regulatory region with a constitutive promoter as described in European Patent Application 0635572 A2, incorporated herein by reference. Production of biotin and biotin vitamers can be further improved by integration and amplification of the deregulated genes in the B. subtilis chromosome. Strain BI282, in European Patent Application 0635572 A2, herein incorporated by reference, is an example of such a strain.
We have found that the conversion of KAPA to DAPA is a serious bottleneck in the biosynthesis of biotin using engineered cells that are fed pimelic acid. As other controls on biotin biosynthesis are removed, the KAPA to DAPA conversion is unable to keep pace with KAPA production, resulting in a build-up of KAPA, without a concomitant increase in the final product. We have also discovered that an important component of the bottleneck is the availability and identity of the amino donor used in the KAPA to DAPA conversion. In general, providing adequate quantities of the amino donor is an important strategy for overcoming the bottleneck. Moreover, a DAPA aminotransferase able to use lysine and related compounds as a source of the amino group to be transfered in the reaction which produces DAPA from KAPA, can significantly improve biosynthetic yields of the downstream biotin vitamers, especially dethiobiotin (DTB).
Although we do not wish to be limited to one specific explanation for our finding to the exclusion of other factors, it appears that providing higher levels of an amino donor which can be used by the available aminotransferase substantially ameliorates the bottleneck discussed above. For example, bacterial production of the biotin vitamers by bacteria whose DAPA aminotransferase uses lysine as an amino donor can be dramatically improved by making sufficient lysine available, either by including it in the fermentation medium or by deregulating the lysine biosynthetic pathway. Such a strategy can also be applied to the use of DAPA aminotransferases of B. subtilis and close relatives, including members of the cluster of Bacillus spp. represented by B. subtilis. The cluster includes, e.g., B. subtilis, B. pumilus, B. licheniformis, B. amyloliquefaciens, B. megaterium, B. cereus and B. thuringiensis. The members of the B. subtilis cluster are genetically and metabolically divergent from the more distantly related Bacillus spp. of clusters represented by B. sphaericus and B. stearothermophilus (Priest, In Bacillus subtilis and Other Gram-Positive Bacteria, supra pp. 3-16, hereby incorporated by reference; Stackebrant, et al. J. Gen. Micro. 133:2523-2529, 1987, hereby incorporated by reference).
Accordingly, one aspect of the invention generally features a method of biosynthesizing (e.g., enzymatically or in fermentations using engineered cells) a biotin vitamer by culturing a bacterium that includes a lysine-utilizing DAPA aminotransferase in an environment enriched in lysine, lysine precursor(s), or analog(s). As used herein, xe2x80x9clysine analogxe2x80x9d means a compound that can serve as an amino donor for a DAPA amino transferase, e.g., (S)-2-aiminoethyl-L-cysteine. The desired biotin vitamer is then recovered from the environment. The ability of an amino donor to be used with a given aminotransferase may be evaluated in any appropriate assay, including but not limited to a bioassay based on that described by Eisenberg and Stoner (1971, infra) in which a DAPA sensitive strain of E. coli is used to measure DAPA aminotransferase activity. Typically, the bacterium will also be deregulated with respect to one or more biotin synthetic pathway steps, e.g., as described in EP 635572, incorporated above. The DAPA aminotransferase may be produced by the cell""s wild-type genetic material, by exogenous nucleic acid introduced into the cell, or both.
As used herein, a xe2x80x9clysine-utilizing DAPA aminotransferasexe2x80x9d means a DAPA aminotransferase capable of converting 8-amino-7-ketopelargonic acid (KAPA) to diaminopelargonic acid (DAPA) utilizing lysine or a compound that is converted to lysine or a compound that can substitute for lysine as the amino donor.
As used herein, an xe2x80x9cenvironment enriched forxe2x80x9d means a bacterial culture in which the concentration of the indicated molecule is greater than that found under standard culture conditions, and greater than is necessary to avoid limiting cell growth in the absence of biotin vitamer overproduction. For example, lysine, a lysine analog, or a lysine precursor may be exogenously added to the culture and totals at least 10 mmoles per liter of culture.
The biotin vitamer product to be recovered and purified can be biotin, dethiobiotin, or diaminopelargonic acid (DAPA). When dethiobiotin or DAPA is recovered, the method may further include the step of converting the recovered dethiobiotin or DAPA to biotin.
In another aspect of the invention a bacterial strain is also engineered to overcome the KAPA-to-DAPA bottleneck by overproducing a DAPA aminotransferase capable of transferring an amino group from an amino donor to 8-amino-7-ketopelargonic acid (KAPA). In a preferred embodiment of this aspect of the invention, the bacterial strain is further engineered to overproduce the biotin vitamer by deregulation of a biotin biosynthetic step other than the KAPA-DAPA step.
To further circumvent the KAPA-to-DAPA bottleneck, the strain may be further engineered to produce multiple DAPA-aminotransferases, relying on different amino donors (e.g., lysine and SAM). These activities may be assayed and distinguished as described in detail below. Briefly, the level of KAPA-to-DAPA conversion may be measured by vitamer bioassays and bioautography of products from bacteria grown in the presence of lysine, methionine, or lysine and methionine.
As used herein, xe2x80x9cSAM-utilizing DAPA amino transferasexe2x80x9d means a DAPA aminotransferase capable of converting 8-amino-7-ketopelargonic acid (KAPA) to diaminopelargonic acid (DAPA) utilizing S-adenosylmethionine (SAM) or a compound that is converted to SAM or a compound that can substitute for SAM as the amino donor. As used herein, xe2x80x9cSAM analogxe2x80x9d means a compound that is structurally similar to SAM that can serve as an amino donor for a DAPA amino transferase.
In other embodiments, methionine and lysine, or their analogs are added to the medium.
One way to provide a lysine-rich environment is to enrich the culture with lysine or a lysine homolog that can donate an amino group to KAPA in the DAPA aminotransferase reaction. Lysine homologs include lysine, (S)-2-aminoethyl-L-cysteine (AEC) and other lysine homologs that can serve as amino donors for a DAPA aminotransferase. Another way to provide a lysine-rich environment is to deregulate the bacterium with respect to lysine production by mutating or engineering it to significantly reduce wild-type control over lysine production. For example, deregulation of a lysine synthetic step includes reducing or removing regulation of transcriptional or other expressional control of a lysine synthetic enzyme, or modification of a lysine synthetic enzyme to reduce or remove control over lysine biosynthesis. Deregulation also includes overproducing compounds which are starting materials in the lysine synthetic pathway, and inhibiting biodegradation of lysine (Amino Acids: Biosynthesis and Genetic Regulations, E. Hermann and R. Somerville (eds.) Addison Wesley, Reading, Mass. 1983, pp. 147-172, 213-244, 417).
Deregulation of a biotin synthetic step includes reducing or removing regulation of transcriptional or other expressional control of a biotin synthetic enzyme, or modification of a biotin synthetic enzyme to reduce or remove control over the enzyme-catalyzed biotin synthetic reaction. It can also include overproducing compounds which are starting materials in the biotin synthetic pathway, and inhibiting biodegradation of a desired biotin vitamer.
Bacteria can be engineered by intentionally and specifically altering the wild-type genome to produce a desired biosynthetic phenotypexe2x80x94e.g., to synthesize more lysine than the corresponding wild-type, unengineered organism, or to remove a bottleneck in the biotin biosynthetic pathway.
Conversion of DTB to biotin may be by any means including but not limited to biochemical conversion of DTB to biotin, feeding DTB to bacteria engineered for the bioconversion of DTB to biotin (Fujisawa et al., Biosci. Biotech. Biochem. 57:740-744, 1993), in vitro synthesis of biotin from DTB (Birch et al., J. Biol. Chem. 270:19158-19165, 1995; Fujisawa et al., FEMS Microbiology Letters 110:1-4, 1993; Ifuku et al., Biosci. Biotech. Biochem. 56:1780-1785, 1992; Birch, WO 94/08023) or chemical synthesis.